Western Blots
Total proteins were extracted with protein extraction buffer (Agrisera)
supplemented with 5 mM DTT and the cocktail of protease inhibitors to
prevent protein degradation. 10 µg of proteins were separated on 15%
acrylamide SDS-PAGE and transferred to 0.45 -µm nitrocellulose membrane
(Amersham 10600003). All proteins apart from WHY1 were detected with
rabbit polyclonal primary antibody (Agrisera) and secondary HRP-linked
anti-rabbit (1:10000, Agrisera AS09 602).
For immunological detection of WHY1, the antibodies were directed toward
the synthetic peptide of recombinant HvWhy1 protein (PRQYDWARKQVF) in
rabbits and antibodies were affinity-purified (Generon, UK). The
specificity of immunodetection was validated using pre-immune sera.